BACTERIA-MEDIATED GENE MODULATION VIA microRNA MACHINERY

ABSTRACT

The present invention provides a method of synthesizing, processing, and/or delivering miRNA or its precursors to eukaryotic cells using bacteria, preferably non-pathogenic or therapeutic strains of bacteria, to effect gene modulation in eukaryotic cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. provisionalpatent application Ser. No. 60/947,311 filed Jun. 29, 2007, the contentof which applications is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

First discovered in Caenorhabditis elegans, microRNA (miRNA) have beenfound in plants and animals including humans. Encoded by genestranscribed from DNA but not translated into protein (non-protein-codingRNA), miRNAs have been found to regulate as much as more than 30%mammalian genes. Mature miRNA molecules are partially complementary toone or more messenger RNA (mRNA) molecules and their main function isbelieved to be modulating gene expression.

With many potential applications of miRNA for therapeutic purposes,however, one major obstacle has been the delivery of miRNA or itsprecursors into target cells. A new method is needed for safe andpredictable administration of miRNAs to animal targets.

SUMMARY OF THE INVENTION

The present invention provides bacteria-mediated system for introducingmiRNA into a target cell through bacterial infection. In one embodiment,the bacterium contains a miRNA-encoding DNA, and expresses the miRNA inthe bacterium through a prokaryotic promoter, or in the target cellusing a eukaryotic promoter. In an alternative embodiment, the bacteriumcontains a DNA that encoding a precursor to the miRNA, and expresses theprecursor in the bacterium through a prokaryotic promoter, or in thetarget cell using a eukaryotic promoter. If the precursor is expressedin the bacterium, it can be processed in the bacterium or in the targetcell into the mature miRNA.

In one aspect, the present invention provides a DNA vector that encodesat least a microRNA (miRNA) or a miRNA precursor. The miRNA is capableof modulating, i.e., up-regulating or down-regulating, the expression ofat least one eukaryotic, prokaryotic, or viral gene. The miRNA precursorcan be a pri-miRNA, a pre-miRNA or a miRNA-duplex. In one embodiment,the vector encodes two miRNAs that have a substantially complementaryregion—when expressed, the two form a duplex. In one embodiment, themiRNA is a mature or guide miRNA. The vector can further include aprokaryotic or eukaryotic promoter, and can further encode an Hly gene.In one embodiment, the at least one gene targeted by the miRNA iscancer-related.

In one aspect, the present invention provides a bacterium that containsa microRNA (miRNA), a miRNA precursor, or a DNA molecule encoding saidmiRNA or said precursor, said miRNA capable of modulating the expressionof at least one eukaryotic, prokaryotic, or viral gene. The bacteriumcan be a live invasive bacterium or a derivate thereof. In oneembodiment, the bacterium further contains an enzyme or ribozyme that iscapable of processing the miRNA precursor closer to a mature miRNA, suchas an endonuclease. In one embodiment, the bacterium further contains atleast one of a bacterial RNase III, a Dicer, a Dicer-like enzyme, Droshaand Pasha. The bacterium can further include an enzyme that assists intransporting its genetic materials, upon their release, into thecytoplasm of the target eukaryotic cell. In one embodiment, that enzymeis listeriolysin O encoded by Hly A gene. The eukaryotic target gene canbe an animal, e.g., mammalian or avian gene. In one embodiment, the atleast one gene targeted by the miRNA is cancer-related.

In one aspect, the present invention provides a method of delivering amiRNA or a miRNA precursor to an animal cell by infecting the animalcell with the bacterium of the present invention. In an embodiment, theanimal cell is a human cell. The method may further include a step oflysing the bacterium after infecting it into the animal cell.

In one aspect, the present invention provides a method of manufacturinga miRNA. In an embodiment, the method includes the step of infecting abacterium with a prokaryotic vector that encodes at least a miRNA.Alternatively, the method includes the step of infecting a bacteriumwith a first prokaryotic vector that encodes at least a miRNA precursor,and a second prokaryotic vector that encodes at least one enzyme forprocessing said miRNA precursor into a miRNA. The method furtherincludes the steps of expressing the miRNA or miRNA precursor and theenzyme, respectively, in the bacterium, and harvesting miRNA from thebacterium.

In one aspect, the present invention provides a method of regulating theexpression of at least one target gene in an animal cell. The methodincludes the steps of: infecting an animal cell with the bacterium ofthe present invention; and lysing said bacterium to release its content,thereby allowing an miRNA from said content or produced from saidcontent to interact with an mRNA of a target gene, and therebyregulating the expression of said gene. In one feature, the mechanism ofregulation is translation repression, mRNA degradation or both.

In one aspect, the present invention provides a method of treating orpreventing a disorder in an animal. The method includes regulating theexpression of at least one target gene known to be involved in thedisorder by infecting the cells of the animal with bacteria comprising amicroRNA (miRNA), a miRNA precursor, or a DNA molecule encoding at leastsaid miRNA or said precursor, said miRNA capable of modulating theexpression of at least said gene. The animal can be mammalian or avian.

In one aspect, the present invention provides a method of treating orpreventing cancer or a cell proliferation disorder in an animal. Themethod includes regulating the expression of at least one target geneknown to be involved in cell proliferation or in cancer by infecting thecells of the animal with bacteria comprising a microRNA (miRNA), a miRNAprecursor, or a DNA molecule encoding at least said miRNA or saidprecursor, said miRNA capable of modulating the expression of at leastsaid gene.

In one aspect, the present invention provides a method of treating orpreventing a disorder in an animal caused by at least one defectivemiRNA in the animal. The method includes infecting the cells of theanimal with bacteria comprising a functional version of said miRNA, aRNA precursor to said functional miRNA, or a DNA molecule encoding atleast said functional miRNA or said precursor.

In one aspect, the present invention provides a method of treating orpreventing a disorder in an animal caused by at least one upregulatedmiRNA in the animal. The method includes infecting the cells of theanimal with bacteria comprising an antisense version of said miRNA, aRNA precursor to said antisense version of the miRNA, or a DNA moleculeencoding at least said antisense version or said precursor.

In another aspect, the present invention provides a method ofdiscovering or validating a therapeutic target. The method includes thesteps of infecting a mammalian cell with the bacterium of the presentinvention; lysing said bacterium to release its content; andinvestigating an interaction between a substrate and an miRNA from saidcontent or produced from said content. The substrate may modulate themiRNA activity or the miRNA may modulate the substrate activity.

Other features and advantages of the present invention are apparent fromthe additional descriptions provided herein including the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing how miRNA is processed from itsvarious precursors.

FIG. 2 is a vector map showing plasmid pTMIR's construction.

FIG. 3 is a vector map showing plasmid pTPIV's construction.

FIG. 4 shows ethidium bromide stained RT-PCR reaction (left) and achemiluminescent western blot (right).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “modulating” refers to either increasing or decreasing(e.g., silencing), in other words, either upregulating ordownregulating. As used herein, “introducing” or “delivering” amicroorganism to a target cell, refers to the process of infecting thetarget cell with the microorganism (e.g., a bacterium), and, in certaincases, releasing the genetic materials inside the microorganism into adesired location of the target cell (e.g., the cytoplasm), possiblythrough lysing the microorganism.

MicroRNAs (miRNAs) are a class of endogenous, single or double-stranded,about 22 nucleotide-long RNA molecules that regulate as much as 30% ofmamalian genes (Czech, NEJM 354:1194-1195 (2006); Mack, Nature Biotech.25:631-638 (2007); Eulalio, et al, Cell 132:9-14 (2008)). miRNArepresses protein production by blocking translation or causingtranscript degradation. An miRNA may target 250-500 different mRNAsmiRNA is the product of the Dicer digestion of pre-miRNA, which in turnis the product of primary miRNA (pri-miRNA).

Dicer is a member of the RNase III ribonuclease family. Dicer cleaveslong double-stranded RNA (dsRNA) and short hairpin RNA (shRNA) intoshort double-stranded RNA fragments called small interfering RNA (siRNA)about 20-25 nucleotides long, usually with a two-base overhang on the 3′end. Dicer also cleaves pre-microRNA (miRNA) into miRNA duplex. Dicercatalyzes the first step in the RNA interference pathway and initiatesformation of the RNA-induced silencing complex (RISC), whose catalyticcomponent argonaute is an endonuclease capable of degrading messengerRNA (mRNA) whose sequence is complementary to that of the siRNA guidestrand.

Referring to FIG. 1, the genes encoding miRNAs are much longer than theprocessed, mature miRNA molecule; miRNAs are first transcribed asprimary transcripts or pri-miRNA with a cap and poly-A tail andprocessed to shorter, 70-nucleotide stem-loop structures known aspre-miRNA in the cell nucleus. This processing is performed in animalsby a protein complex known as the Microprocessor complex, consisting ofthe nuclease Drosha and the double-stranded RNA binding protein Pasha.These pre-miRNAs are then processed to mature miRNAs in the cytoplasm byinteraction with the endonuclease Dicer, which also initiates theformation of the RNA-induced silencing complex (RISC). This complex isresponsible for the gene silencing observed due to miRNA expression andRNA interference. The pathway is also different for miRNAs derived fromintronic stem-loops; these are processed by Dicer but not by Drosha.Either the sense strand or antisense strand of DNA can function astemplates to give rise to miRNA.

As shown in FIG. 1, there are at least three forms of intermediate RNAprecursors to a mature miRNA: (a) pri-miRNA, (b) pre-miRNA and (c) amiRNA duplex that results from Dicer cleavage of the stem-loop structureof pre-miRNA.

Accordingly, in one aspect, the present invention provides a system thatdelivers miRNA, or miRNA precursor, or a DNA encoding the miRNA or miRNAprecursor, or a mixture of any of the above to target cells usingbacteria, to effect gene modulation through either translationrepression, mRNA degradation or both. The bacteria is preferablynon-pathogenic or therapeutic strains with the capability to entercells. The target cells can be eukaryotic cells. The miRNA of thepresent invention modulates, e.g., downregulates, genes of interest intarget cells. The eukaryotic cells can be mammalian cells or aviancells. The gene of interest can be a mammalian, avian, eukaryotic,bacterial or viral gene. If the molecule delivered is a miRNA precursor,it can be processed into a mature miRNA inside bacteria, or in thetarget cell using cell's existing processing machinery. In animal cells,enzymes or ribozymes such as Drosha, Pasha, and Dicer, are part of thismachinery, which processes the miRNA precursors into a mature form thatcan guide the multi-enzyme complex, RNA-induced silencing complex(RISC), to the target mRNA.

In one embodiment, the present invention provides a prokaryotic vectorthat encodes at least a miRNA or a miRNA precursor. The miRNA canmodulate the expression of at least one eukaryotic, prokaryotic, orviral gene. The miRNA precursor can be (a) pri-miRNA, (b) pre-miRNA or(c) a miRNA duplex. For the miRNA duplex, the vector, which can be adouble-strand circular plasmid, encodes two miRNAs that have acomplementary region to form the duplex. The plasmid can have one or twopromotors. The promoter can be prokaryotic or eukaryotic depending onwhere the vector is to be expressed. In one embodiment, at least oneprokaryotic promoter, such as T7, is provided on the vector as thevector is intended to be expressed inside a carrier bacterium. Inanother embodiment, at least one eurkaryotic promoter is provided on thevector as the vector is intended to be expressed inside the targeteukaryotic cell.

In one embodiment, one or more DNA molecules encoding the miRNAprecursor carried by the bacteria is delivered to the eukaryotic cells,transcribed in the eukaryotic cells and then processed to produce amature miRNA in the eukaryotic cells. The DNA molecules can be under thecontrol of RNA-polymerase II compatible promoters, or RNA-polymerase IIIcompatible promoters (e.g., U6, H1), which usually direct thetranscription of small nuclear RNAs (snRNAs) (P. J. Paddison, A. A.Caudiy, G. J. Harmon, PNAS 99, 1443 (2002), T. R. Brummelkamp, R.Bernards, R. Agami, Science 296, 550 (2002)). A double “Trojan horse”technique can be used with an invasive and auxotrophic bacteriumcarrying a eukaryotic transcription plasmid. This plasmid is, in turn,transcribed by the target cell to form a miRNA precursor which isfurther processed into a mature miRNA that triggers the intracellularprocess of RNAi.

In one advantageous aspect, the present invention provides a method ofdelivering miRNAs, miRNA precursors, or DNA encoding the miRNAs orprecursors to eukaryotic cells by infecting the cells with bacteria,preferably non-pathogenic or therapeutic strains of bacteria, to effectgene modulation in eukaryotic cells. The bacteria comprise (a) a miRNA,(b) a miRNA precursor, or (c) a DNA encoding the miRNA or the precursor.

In an embodiment, the bacterium itself is capable of synthesizing andprocessing the miRNA precursor into a mature miRNA. In one embodiment,the bacterium has an enzyme or ribozyme to process or digest theprecursors. The enzyme can be an endonuclease. The endonuclease can be amember of RNase III family, such as a bacterial RNase III, a Dicer, orDicer-like enzyme. The enzyme may be Drosha or Pasha. In one embodiment,the enzyme is endogenous to the carrier bacterium. In anotherembodiment, the enzyme is exogenous to the carrier bacterium andintroduced through vectors that express such enzyme, e.g., a dicer-likeenzyme, or a Drosha-like enzyme.

The resulting miRNA can modulate, e.g., knockdown or silence, theexpression of one or more target genes, which means it is capable ofreducing the expression of the gene by at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or more than 90%.

Bacterial delivery is more attractive than viral delivery as it can becontrolled by use of antibiotics and attenuated bacterial strains whichare unable to multiply. Also, bacteria are much more accessible togenetic manipulation which allows the production of vector strainsspecifically tailored to certain applications. In one embodiment of theinvention, the methods of the present invention are used to createbacteria that cause gene modulation in a tissue specific manner.

The non-virulent bacteria of the present invention may enter a mammalianhost cell through various mechanisms. Professional phagocytes activelyengulfing bacteria, invasive bacteria strains have the ability to invadenon-phagocytic host cells. Naturally occurring examples of such bacteriaare intracellular pathogens such as Listeria, Shigella and Salmonella,but this property can also be transferred to other bacteria such as E.coli and Bifidobacteriae, including probiotics through transfer ofinvasion-related genes (P. Courvalin, S. Goussard, C. Grillot-Courvalin,C. R. Acad. Sci. Paris 318, 1207 (1995)). In other embodiments of theinvention, bacteria used to deliver small RNAs to host cells includeShigella flexneri (D. R. Sizemore, A. A. Branstrom, J. C. Sadoff,Science 270, 299 (1995)), invasive E. coli (P. Courvalin, S. Goussard,C. Grillot-Courvalin, C. R. Acad. Sci. Paris 318,1207 (1995), C.Grillot-Courvalin, S. Goussard, F. Huetz, D. M. Ojcius, P. Courvalin,Nat Biotechnol 16, 862 (1998)), Yersinia enterocolitica (A. Al-Marini A,A. Tibor, P. Lestrate, P. Mertens, X. De Bolle, J. J. Letesson InfectImmim 70, 1915 (2002)) and Listeria monocytogenes (M. Hense, E. Domann,S. Krusch, P. Wachholz, K. E. Dittmar, M. Rohde, J. Wehland, T.Chakraborty, S. Weiss, Cell Microbial 3, 599 (2001), S. Pilgrim, J.Stritzker, C. Schoen, A. Kolb-Maurer, G. Geginat, M. J. Loessner, I.Gentschev, W. Goebel, Gene Therapy 10, 2036 (2003)). Any invasivebacterium is useful for DNA transfer into eukaryotic cells (S. Weiss, T.Chakraborty, Curr Opinion Biotechnol 12, 467 (2001)).

In an embodiment, a prokaryotic vector that encodes at least a microRNA(miRNA) or a miRNA precursor is constructed. The miRNA can modulate theexpression of at least one eukaryotic, prokaryotic, or viral gene. Thenan invasive bacterium is transformed with the vector. The bacterium canbe a live one, or a derivate thereof, e.g., a half-dead bacterium orfunctional particle derived from the bacterium. In one embodiment, thebacterium is capable of expressing and/or processing the miRNA or itsprecursor. In an alternative embodiment, the bacterium is not capable ofexpressing and/or processing the miRNA or its precursor, but only actsas a carrier for further expression and processing to take place in thetarget eukaryotic cell. The miRNA precursor can be (a) pri-miRNA, (b)pre-miRNA, (c) a miRNA duplex, or a mixture thereof.

At this point, the transformed bacterium contains one or more of thefollowing: a microRNA (miRNA), a miRNA precursor, a DNA moleculeencoding said miRNA or said precursor, or a mixture of any of the above.

The content of the bacterium is delivered to the target cell viabacterial invasion (“bactofection”), and is liberated within themammalian target cell after bacterial lysis triggered either byauxotrophy, endosomes, or by timed addition of antibiotics, resulting inmodulation of the target genes.

Liberation of bacterial DNAs and RNAs from the intracellular bacteriamay occur through active mechanisms. The bacterial DNAs and RNAs maycomprise a mixture of miRNAs, miRNA precursors, and/or their encodingplasmids. One mechanism involves the type III export system in S.typhimurium, a specialized multiprotein complex spanning the bacterialcell membrane whose functions include secretion of virulence factors tothe outside of the cell to allow signaling towards the target cell, butwhich can also be used to deliver antigens into target cells. (RüssmannH. Int J Med Microbiol, 293:107-12 (2003)) or through bacterial lysisand liberation of bacterial contents into the cytoplasm. The lysis ofintracellular bacteria is triggered through addition of anintracellularly active antibiotic (tetracycline) or occurs naturallythrough bacterial metabolic attenuation (auxotrophy) or through cellularendosome or lysosome. After liberation of the eukaryotic transcriptionplasmid, miRNAs, miRNA precursors, are produced within the target celland, in turn, trigger the miRNA-based modulation of the targetedgene(s).

The present invention can be performed using the naturally invasivepathogen Salmonella typhimurium. In one aspect of this embodiment, thestrains of Salmonella typhimurium include SL 7207 and VNP20009 (S. K.Hoiseth, B. A. D. Stacker, Nature 291, 238 (1981); Pawelek J M, Low K B,Bermudes D. Cancer Res. 57(20):4537-44 (Oct. 15, 1997)).

In another embodiment of the invention, the present invention isperformed using attenuated E. coli. In one example of this embodiment,the strain of E. coli is BM 2710 (C. Grillot-Courvalin, S. Goussard, F.Huetz, D. M. Ojcius, P. Courvalin, Nat Biotechnol 16, 862 (1998)). Inone feature of this embodiment, the BM 2710 strain is engineered topossess cell-invading properties through an invasion plasmid, e.g., onethat encodes the Inv gene. According to another feature of the presentinvention, the bacterium of the invention contains a vector that has theHlyA (listeriolysine O) gene, as the Hly protein is considered importantfor genetic materials escape from the entry vesicles. Obviously, thatvector could be the same invasion plasmid. Accordingly, in oneembodiment, the bacterium has a plasmid that encodes both the Inv andHly genes. Accordingly, in one embodiment, the bacterium has a plasmidthat encodes both the Inv and Hly genes. In one aspect of the invention,this plasmid is pGB2inv-hly. In one example, the E. coli strain used inthe present invention is BL21 (DE3) pLysE.

In an embodiment, a miRNA precursor, e.g., a pre-miRNA, is expressed andprocessed into miRNA in the bacteria and then delivered into theeukaryotic cells. In another embodiment, a miRNA precursor, e.g., apre-miRNA, is expressed in the bacteria and then delivered into theeukaryotic cells and processed into miRNA in the eukaryotic cells. In analternative embodiment, a DNA molecule encoding a miRNA precursor, e.g.,a pri-miRNA, is delivered into the eukaryotic cells, and the miRNAprecursor is expressed and then processed into miRNA in the eukaryoticcells.

In an embodiment, a miRNA is expressed in the bacteria and thendelivered into the eukaryotic cells. In another embodiment, a DNAmolecule encoding a miRNA is delivered into the eukaryotic cells, andthe miRNA is expressed in the eukaryotic cells.

In an embodiment, the at least one gene is one gene, or more than 2, 4,8, 16, 32, 64, 100, 200, or 400 genes.

The present invention also provides a method to manufacture microRNA bytransforming a bacterium or other host cell with a vector that expresseseither the miRNA or a precursor under the control of an appropriatepromoter. In the case of bacteria, a prokaryotic promoter, e.g., T7, isprovided in the vector. If the vector encodes a miRNA precursor, e.g., apre-miRNA, vectors that express needed processing enzymes discussedherein above are also transformed into the bacterium.

1. Applications 1.1. Research and Drug Discovery Tools

Wildtype miRNAs serve gene-regulatory functions in cells; some have beenassociated with various types of human diseases, including cancers.Therefore, miRNAs can be used to study their regulatory target genes andhow they themselves are targeted by other molecules, e.g, miRNAinhibitors.

Methods of the invention can be used to study gene function in vitro andin vivo. Vectors of the invention can be used to transfect culturedanimal cells as a research tool in drug target/pathway identificationand validation. For example, after infecting the host cells with thebacteria of the invention and releasing the bacterial content to providecertain miRNA or precursor that can be processed into miRNA, cells canbe observed for phenotypical or morphology changes that suggest somepathway of interest has been affected. Such phenotypical changes caninvolve numbers of nuclei, nuclei morphology, cell death, cellproliferation, DNA fragmentation, cell surface marker, and mitoticindex, etc. In another example, interaction between a molecule/substratein the host cell and the miRNA transfected into the cell can be isolatedor identified to discover potential therapeutic target. That target canbe upstream and modulates the miRNA activity, or, the target can bedownstream and its activity is modulated by the miRNA.

In terms of in vivo applications, since miRNA can be introduced into ahost body by the method of the present invention, a systems biologyapproach can be adopted in studying the effect of certain miRNA ondifferent cell types and different tissues.

These in vivo and in vitro methods use bacteria with desirableproperties (invasiveness, attenuation, steerability). For example,Bifidobacteria and Listeria, are used to perform bacteria-mediated RNAimethods of the present invention. Invasiveness as well as eukaryotic orprokaryotic transcription of one or several miRNAs or miRNA precursorsis conferred to a bacterium using plasmids.

1.2 Therapeutic Uses

The bacteria-mediated miRNA compositions of the present invention can beused for the treatment and or prevention of various diseases, includingthe diseases summarized in Dykxhoorn, Novina & Sharp. Nat. Rev. Mol.Cell. Biol. 4:457-467 (2003); Kim & Rossi, Nature Rev. Genet. 8:173-184(2007); de Fougerolles, et al. Nature Rev. Drug Discov. 6:443-453(2007); Czech, NEJM 354:1194-1195 (2006); and Mack, Nature Biotech.25:631-638 (2007).

In an embodiment, the present invention can be used as a cancer therapyor to prevent cancer by targeting one or more cancer-related gene. Thismethod is effected by silencing or knockingdown genes involved with cellproliferation or other cancer phenotypes. The bacteria of the presentinvention used for cancer treatment is preferably bacteria engineered tosafely seek out and kill tumors (Forbes, Nature Biotechnology24:1484-1485 (2006)). The bacteria can be an obligate anaerobe, such asClostridium novyi-NT, or facultative anaerobe, such as Salmonellatyphimurium and Escherichia coli. (ibid)

Examples of these genes are various oncogenes, such as k-Ras. Forexample, k-Ras has been shown to be regulated by miRNA Let-7. Theseoncogenes are active and relevant in the majority of clinical cases. Forexample, K-Ras is aberrantly active in the majority of human coloncancer, pancreatic cancer, and non small cell lung cancers. K-Rasmutation confers resistance to chemotherapy and current targetedtherapy. The bacteria-mediated and miRNA-based gene targeting methods ofthe present invention can be applied to reach the intestinal tract forcolon cancer treatment and prevention. These methods are also used totreat animals carrying xenograft tumors, to treat and prevent cancer ink-RasV12 model of intestinal tumorgenesis, and to prevent and treattumors in the adenomatous polyposis coli min mouse model (APC-minmodel).

The methods of the present invention can also be used to createcancer-preventing “probiotic bacteria” for use, especially with thetarget of GI tract or liver. The methods of the present invention areused as therapy against inflammatory conditions, e.g. inflammatory boweldisease (IBD), including crohn's diseases, ulcerative colitis. Thesemethods are used to silence or knockdown non-cancer gene targets (viralgenes, for treatment and prevention of hepatitis B, C; inflammatorygenes, for treatment and prevention of inflammatory bowel disease) andothers.

The methods of the present invention can be used for delivery of genesilencing to the gut and colon, and for oral application in thetreatment of various diseases, namely colon cancer treatment andprevention. In another aspect of this embodiment, delivery of genesilencing is extra-intestinal, such as topical, intravenous.

These bacteria produced and/or delivered miRNA molecules can also beused to silence or knockdown non-cancer gene targets. The RNA moleculesof the invention can also be used to treat or prevent ocular diseases,(e.g., age-related macular degeneration (AMD) and diabetic retinopathy(DR)); infectious diseases (e.g. HIV/AIDS, hepatitis B virus (HBV),hepatitis C virus (HCV), human papillomavirus (HPV), herpes simplexvirus (HSV), RCV, cytomegalovirus (CMV), dengue fever, west Nile virus);respiratory diseases (e.g., respiratory syncytial virus (RSV), asthma,cystic fibrosis); neurological diseases (e.g., Huntingdon's disease(HD), amyotrophic lateral sclerosis (ALS), spinal cord injury,Parkinson's disease, Alzheimer's disease, pain); cardiovasculardiseases; metabolic disorders (e.g., diabetes); genetic disorders; andinflammatory conditions (e.g., inflammatory bowel disease (IBD),arthritis, rheumatoid disease, autoimmune disorders), dermatologicaldiseases.

Because miRNAs are part of animal cell's regulatory mechanism of manycellular pathways, defective miRNA itself may cause disorders ordiseases in animals. Therefore, one therapeutic use of the presentinvention is simply producing and delivering functional copies orgenetically engineered copies of miRNA into an animal to treat orprevent such disorder or disease.

2. Bacteria Delivery

According to the invention, any microorganism which is capable ofdelivering a molecule, e.g., a miRNA molecule, into the cytoplasm of atarget cell, such as by traversing the membrane and entering thecytoplasm of a cell, can be used to deliver miRNA and its precursors tosuch cells. In a preferred embodiment, the microorganism is aprokaryote. In an even more preferred embodiment, the prokaryote is abacterium. Also within the scope of the present invention aremicroorganisms other than bacteria which can be used for delivering RNAto a cell. For example, the microorganism can be a fungus, e.g.,Cryptococciis neoformans, protozoan, e.g., Trypanosoma cruzi, Toxoplasmagondii, Leishmania donovani, and plasmodia.

As used herein, the term “invasive” when referring to a microorganism,e.g., a bacterium, refers to a microorganism which is capable ofdelivering at least one molecule, e.g., an RNA or RNA-encoding DNAmolecule, to a target cell. An invasive microorganism can be amicroorganism which is capable of traversing a cell membrane, therebyentering the cytoplasm of said cell, and delivering at least some of itscontent, e.g., RNA or RNA-encoding DNA, into the target cell. Theprocess of delivery of the at least one molecule into the target cellpreferably does not significantly modify the invasion apparatus. In apreferred embodiment, the microorganism is a bacterium. A preferredinvasive bacterium is a bacterium which is capable of delivering atleast one molecule, e.g., an RNA or RNA-encoding DNA molecule, to atarget cells, such as by entering the cytoplasm of a eukaryotic cell.Preferred invasive bacteria are live bacteria, e.g., live invasivebacteria. Invasive microorganisms include microorganisms that arenaturally capable of delivering at least one molecule to a target cell,such as by traversing the cell membrane, e.g., a eukaryotic cellmembrane, and entering the cytoplasm, as well as microorganisms whichare not naturally invasive and which have been modified, e.g.,genetically modified, to be invasive. In another preferred embodiment, amicroorganism which is not naturally invasive can be modified to becomeinvasive by linking the bacterium to an “invasion factor”, also termed“entry factor” or “cytoplasm-targeting factor”. As used herein, an“invasion factor” is a factor, e.g., a protein or a group of proteinswhich, when expressed by a non-invasive bacterium, render the bacteriuminvasive. As used herein, an “invasion factor” is encoded by a“cytoplasm-targeting gene”. Naturally invasive microorganisms, e.g.,bacteria, may have a certain tropism, i.e., preferred target cells.Alternatively, microorganisms, e.g., bacteria can be modified, e.g.,genetically, to mimic the tropism of a second microorganism.

Delivery of at least one molecule into a target cell can be determinedaccording to methods known in the art. For example, the presence of themolecule, by the decrease in expression of an RNA or protein silencedthereby, can be detected by hybridization or PCR methods, or byimmunological methods which may include the use of an antibody.Determining whether a microorganism is sufficiently invasive for use inthe present invention may include determining whether sufficient RNA(miRNA or miRNA precursors) or its encoding DNA, was delivered to hostcells, relative to the number of microorganisms contacted with the hostcells. If the amount of RNA, is low relative to the number ofmicroorganisms used, it may be desirable to further modify themicroorganism to increase its invasive potential.

Bacterial entry into cells can be measured by various methods.Intracellular bacteria survive treatment by aminoglycoside antibiotics,whereas extracellular bacteria are rapidly killed. A quantitativeestimate of bacterial uptake can be achieved by treating cell monolayerswith the antibiotic gentamicin to inactivate extracellular bacteria,then by removing said antibiotic before liberating the survivingintracellular organisms with gentle detergent and determining viablecounts on standard bacteriological medium. Furthermore, bacterial entryinto cells can be directly observed, e.g., by thin-section-transmissionelectron microscopy of cell layers or by immunofluorescent techniques(Falkow et al. (1992) Annual Rev. Cell Biol. 8:333). Thus, varioustechniques can be used to determine whether a specific bacteria iscapable of invading a specific type of cell or to confirm bacterialinvasion following modification of the bacteria, such modification ofthe tropism of the bacteria to mimic that of a second bacterium.Bacteria that can be used for delivering RNA according to the method ofthe present invention are preferably non-pathogenic. However, pathogenicbacteria can also be used, so long as their pathogenicity has beenattenuated, to thereby render the bacteria non-harmful to a subject towhich it is administered. As used herein, the term “attenuatedbacterium” refers to a bacterium that has been modified to significantlyreduce or eliminate its harmfulness to a subject. A pathogenic bacteriumcan be attenuated by various methods, set forth below.

Without wanting to be limited to a specific mechanism of action, thebacterium delivering the RNA or DNA into the eukaryotic cell can entervarious compartments of the cell, depending on the type of bacterium.For example, the bacterium can be in a vesicle, e.g., a phagocyticvesicle. Once inside the cell, the bacterium can be destroyed or lysedand its contents delivered to the eukaryotic cell. A bacterium can alsobe engineered to express a phago some degrading enzyme to allow leakageof RNA from the phagosome. In some embodiments, the bacterium can stayalive for various times in the eukaryotic cell and may continue toproduce RNA (miRNA or miRNA precursors). The RNA or RNA-encoding DNA canthen be released from the bacterium into the cell by, e.g., leakage. Incertain embodiments of the invention, the bacterium can also replicatein the eukaryotic cell. In a preferred embodiment, bacterial replicationdoes not kill the host cell. The present invention is not limited todelivery of RNA or RNA-encoding DNA by a specific mechanism and isintended to encompass methods and compositions permitting delivery ofRNA or RNA-encoding DNA by a bacterium independently of the mechanism ofdelivery.

Set forth below are examples of bacteria which have been described inthe literature as being naturally invasive (section 2.1), as well asbacteria which have been described in the literature as being naturallynon-invasive bacteria (section 2.2), as well as bacteria which arenaturally non-pathogenic or which are attenuated. Although some bacteriahave been described as being non-invasive (section 2.2), these may stillbe sufficiently invasive for use according to the invention. Whethertraditionally described as naturally invasive or noninvasive, anybacterial strain can be modified to modulate, in particular to increase,its invasive characteristics (e.g., as described in section 2.3).

2.1 Naturally Invasive Bacteria

The particular naturally invasive bacteria employed in the presentinvention is not critical thereto. Examples of such naturally-occurringinvasive bacteria include, but are not limited to, Shigella spp.,Salmonella spp., Listeria spp., Rickettsia spp., and enteroinvasiveEscherichia coli. The particular Shigella strain employed is notcritical to the present invention.

Examples of Shigella strains which can be employed in the presentinvention include Shigella flexneri 2a (ATCC No. 29903), Shigella sonnet(ATCC No. 29930), and Shigella disenteriae(ATCC No. 13313). Anattenuated Shigella strain, such as Shigella flexneri 2a 2457T aroA virGmutant CVD 1203 (Noriega et al. supra), Shigella flexneri M90T icsAmutant (Goldberg et al Infect Immun., 62:5664-5668 (1994)), Shigellaflexneri Y SFL1 14 aroD mutant (Karnen et al. Vacc, 10:167-174 (1992)),and Shigella flexneri aroA aroD mutant (Verma et al Vacc, 9:6-9 (1991))are preferably employed in the present invention. Alternatively, newattenuated Shigella spp. strains can be constructed by introducing anattenuating mutation either singularly or in conjunction with one ormore additional attenuating mutations.

At least one advantage to Shigella RNA vaccine vectors is their tropismfor lymphoid tissue in the colonic mucosal surface. In addition, theprimary site of Shigella replication is believed to be within dendriticcells and macrophages, which are commonly found at the basal lateralsurface of M cells in mucosal lymphoid tissues (reviewed by McGhee, J.R. et al Reproduction, Fertility, & Development 6:369 (1994); Pascual,D. W. et al Immunomethods 5:56 (1994)). As such, Shigella vectors mayprovide a means to express antigens in these professional antigenpresenting cells. Another advantage of Shigella vectors is thatattenuated Shigella strains deliver nucleic acid reporter genes in vitroand in vivo (Sizemore, D. R. et al. Science 270:299 (1995); Courvalin,P. et al Comptes Rendus de I Academie des Sciences Serie Ill-Sciences deIa Vie-Life Sciences 318:1207 (1995); Powell, R. J. et al In: Molecularapproaches to the control of infectious diseases (1996). F. Brown, E.Norrby, D. Burton and J. Mekalanos, eds. Cold Spring Harbor LaboratoryPress, New York. 183; Anderson, R. J. et al Abstracts for the 97thGeneral Meeting of the American Society for Microbiology: E. (1997)). Onthe practical side, the tightly restricted host specificity of Shigellastands to prevent the spread of Shigella vectors into the food chain viaintermediate hosts. Furthermore, attenuated strains that are highlyattenuated in rodents, primates and volunteers have been developed(Anderson et al (1997) supra; Li, A. et al Vaccine 10:395 (1992); Li, A.et al Vaccine 11:180 (1993); Karnell, A. et al Vaccine 13:88 (1995);Sansonetti, P. J. and J. Arondel Vaccine 7:443 (1989); Fontaine, A. etal. Research in Microbiology 141:907 (1990); Sansonetti, P. J. et al.(1991) Vaccine 9:416; Noriega, F. R. et al. Infection & Immunity 62:5168(1994); Noriega, F. R. et al. Infection & Immunity 64:3055 (1996);Noriega, F. R. et al. Infection & Immunity 64:23 (1996); Noriega, F. R.et al. Infection & Immunity 64:3055 (1996); Kotloff, K. L. et al.Infection & Immunity 64:4542 (1996)). This latter knowledge will allowthe development of well tolerated Shigella vectors for use in humans.

Attenuating mutations can be introduced into bacterial pathogens usingnon-specific mutagenesis either chemically, using agents such asN-methyl-N′-nitro-N-nitrosoguanidine, or using recombinant DNAtechniques; classic genetic techniques, such as Tn10 mutagenesis,P22-mediated transduction, λ phage mediated crossover, and conjugationaltransfer; or site-directed mutagenesis using recombinant DNA techniques.Recombinant DNA techniques are preferable since strains constructed byrecombinant DNA techniques are far more defined. Examples of suchattenuating mutations include, but are not limited to: (i) auxotrophicmutations, such as aro (Hoiseth et al. Nature, 291:238-239 (1981)), gua(McFarland et al Microbiol. Path., 3:129-141 (1987)), nad (Park et al.J. Bact, 170:3725-3730 (1988), thy (Nnalue et al. Infect. Immun.,55:955-962 (1987)), and asd (Curtiss, supra) mutations;

(ii) mutations that inactivate global regulatory functions, such as cya(Curtiss et al. Infect. Immun., 55:3035-3043 (1987)), crp (Curtiss et al(1987), supra), phoP/phoQ (Groisman et al. Proc. Natl. Acad. Sci., USA,86:7077-7081 (1989); and Miller et al. Proc. Natl. Acad. Sci., USA,86:5054-5058 (1989)), phop^(c) (Miller et al. J. Bact, 172:2485-2490(1990)) or ompR (Dorman et al. Infect. Immun., 57:2136-2140 (1989))mutations;

(iii) mutations that modify the stress response, such as recA (Buchmeieret al. MoI. Micro., 7:933-936 (1993)), htrA (Johnson et al. Mol. Micro.,5:401-407 (1991)), htpR (Neidhardt et al. Biochem. Biophys. Res. Corn.,100:894-900 (1981)), hsp (Neidhardt et al. Ann. Rev. Genet, 18:295-329(1984)) and groEL (Buchmeier et al. Sci., 248:730-732 (1990)) mutations;

(iv) mutations in specific virulence factors, such as IsyA (Libby et al.Proc. Natl. Acad. Sci., USA, 91:489-493 (1994)), pag or prg (Miller etal (1990), supra; and Miller et al (1989), supra), iscA or virG(d'Hauteville et al. Mol. Micro., 6:833-841 (1992)), plcA

(Mengaud et al. Mol. Microbiol., 5:367-72 (1991); Camilli et al. J. Exp.Med, 173:751-754 (1991)), and act (Brundage et al. Proc. Natl. Acad.Sci., USA, 90:11890-11894 (1993)) mutations; (v) mutations that affectDNA topology, such as top A (Galan et al. Infect. Immun., 58: 1879-1885(1990));

(vi) mutations that disrupt or modify the cell cycle, such as min (deBoer et al. Cell, 56:641-649 (1989)).

(vii) introduction of a gene encoding a suicide system, such as sacB(Recorbet et al. App. Environ. Micro., 59:1361-1366 (1993); Quandt etal. Gene, 127:15-21 (1993)), nuc (Ahrenholtz et al. App. Environ.Micro., 60:3746-3751 (1994)), hok, gef, kil, or phiA (Molin et al. Ann.Rev. Microbiol., 47:139-166 (1993));

(viii) mutations that alter the biogenesis of lipopolysaccharide and/orlipid A, such as rFb (Raetz in Esherishia coli and Salmonellatyphimurium, Neidhardt et al, Ed., ASM Press, Washington D.C. pp1035-1063 (1996)), galE (Hone et al. J. Infect. Dis., 156:164-167(1987)) and htrB (Raetz, supra), msbB (Reatz, supra)

(ix) introduction of a bacteriophage lysis system, such as lysogensencoded by P22 (Rennell et al. Virol, 143:280-289 (1985)), mureintransglycosylase (Bienkowska-Szewczyk et al. Mol. Gen. Genet.,184:111-114 (1981)) or S-gene (Reader et al. Virol, 43:623-628 (1971));and

The attenuating mutations can be either constitutively expressed orunder the control of inducible promoters, such as the temperaturesensitive heat shock family of promoters (Neidhardt et al. supra), orthe anaerobically induced nirB promoter (Harbome et al. Mol. Micro.,6:2805-2813 (1992)) or repressible promoters, such as uapA (Gorfinkielet al. J. Biol. Chem., 268:23376-23381 (1993)) or gcv (Stauffer et al.J. Bact, 176:6159-6164 (1994)).

The particular Listeria strain employed is not critical to the presentinvention. Examples of Listeria strains which can be employed in thepresent invention include Listeria monocytogenes (ATCC No. 15313).Attenuated Listeria strains, such as L. monocytogenes actA mutant(Brundage et al. supra) or L. monocytogenes picA (Camilli et al. J. Exp.Med., 173:751-754 (1991)) are preferably used in the present invention.Alternatively, new attenuated Listeria strains can be constructed byintroducing one or more attenuating mutations in groups (i) to (vii) asdescribed for Shigella spp. above. The particular Salmonella strainemployed is not critical to the present invention.

Examples of Salmonella strains which can be employed in the presentinvention include Salmonella typhi (ATCC No. 7251) and S. typhimurium(ATCC No. 13311). Attenuated Salmonella strains are preferably used inthe present invention and include S. typhi-aroC-aroD (Hone et al. Vacc.9:810 (1991) and S. typhiinurium-axoA mutant (Mastroeni et al. Micro.Pathol. 13:477 (1992)). Alternatively, new attenuated Salmonella strainscan be constructed by introducing one or more attenuating mutations asdescribed fro Shigella spp. above.

The particular Rickettsia strain employed is not critical to the presentinvention. Examples of Rickettsia strains which can be employed in thepresent invention include Rickettsia Rickettsiae (ATCC Nos. VR149 andVR891), Ricketsia prowaseckii (ATCC No. VR233), Rickettsia tsutsugamuchi(ATCC Nos. VR312, VR150 and VR609), Rickettsia mooseri (ATCC No. VR144),Rickettsia sibirica (ATCC No. VR151), and Rochalimaea quitana (ATCC No.VR358). Attenuated Rickettsia strains are preferably used in the presentinvention and can be constructed by introducing one or more attenuatingmutations in groups (i) to (vii) as described for Shigella spp. above.

The particular enteroinvasive Escherichia strain employed is notcritical to the present invention. Examples of enteroinvasiveEscherichia strains which can be employed in the present inventioninclude Escherichia coli strains 4608-58, 1184-68, 53638-C-17, 13-80,and 6-81 (Sansonetti et al. Ann. Microbiol. (Inst. Pasteur),132A:351-355 (1982)).

Attenuated enteroinvasive Escherichia strains are preferably used in thepresent invention and can be constructed by introducing one or moreattenuating mutations in groups (i) to (vii) as described for Shigellaspp. above.

Furthermore, since certain microorganisms other than bacteria can alsointeract with integrin molecules (which are receptors for certaininvasion factors) for cellular uptake, such microorganisms can also beused for introducing RNA into target cells. For example, viruses, e.g.,foot-and-mouth disease virus, echovirus, and adenovirus, and eukaryoticpathogens, e.g., Histoplasma capsulatum and Leishmania major interactwith integrin molecules.

2.2 Less Invasive Bacteria

Examples of bacteria which can be used in the present invention andwhich have been described in the literature as being non-invasive or atleast less invasive than the bacteria listed in the previous section(2.1) include, but are not limited to, Yersinia spp., Escherichia spp.,Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp.,Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydiaspp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionellaspp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibriospp., Bacillus spp., and Erysipelothrix spp. It may be necessary tomodify these bacteria to increase their invasive potential. Theparticular Yersinia strain employed is not critical to the presentinvention.

Examples of Yersinia strains which can be employed in the presentinvention include Y. enterocolitica (ATCC No. 9610) or Y. pestis (ATCCNo. 19428). Attenuated Yersinia strains, such as Y. enterocoliticaYeO3-R2 (al-Hendy et al. Infect. Immun., 60:870-875 (1992)) or Y.enterocolitica aroA (O'Gaora et al. Micro. Path., 9:105-116 (1990)) arepreferably used in the present invention. Alternatively, new attenuatedYersinia strains can be constructed by introducing one or moreattenuating mutations in groups (i) to (vii) as described for Shigellaspp. above.

The particular Escherichia strain employed is not critical to thepresent invention. Examples of Escherichia strains which can be employedin the present invention include E. coli H10407 (Elinghorst et alInfect. Immun., 60:2409-2417 (1992)), and E. coli EFC4, CFT325 andCPZ005 (Donnenberg et al. J. Infect. Dis., 169:831-838 (1994)).Attenuated

Escherichia strains, such as the attenuated turkey pathogen E. coli 02carAB mutant (Kwaga et al. Infect. Immun., 62:3766-3772 (1994)) arepreferably used in the present invention. Alternatively, new attenuatedEscherichia strains can be constructed by introducing one or moreattenuating mutations in groups (i) to (vii) as described for Shigellaspp. above.

The particular Klebsiella strain employed is not critical to the presentinvention.

Examples of Klebsiella strains which can be employed in the presentinvention include K. pneumoniae (ATCC No. 13884). Attenuated Klebsiellastrains are preferably used in the present invention, and can beconstructed by introducing one or more attenuating mutations in groups(i) to (vii) as described for Shigella spp. above.

The particular Bordetella strain employed is not critical to the presentinvention.

Examples of Bordetella strains which can be employed in the presentinvention include B. bronchiseptica (ATCC No. 19395). AttenuatedBordetella strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Neisseria strain employed is not critical to the presentinvention. Examples of Neisseria strains which can be employed in thepresent invention include N. meningitidis (ATCC No. 13077) and N.gonorrhoeae (ATCC No. 19424). Attenuated Neisseria strains, such as N.gonorrhoeae MS11 aro mutant (Chamberlain et al. Micro. Path., 15:51-63(1993)) are preferably used in the present invention. Alternatively, newattenuated Neisseria strains can be constructed by introducing one ormore attenuating mutations in groups (i) to (vii) as described forShigella spp. above. The particular Aeromonas strain employed is notcritical to the present invention. Examples of Aeromonas strains whichcan be employed in the present invention include A. eucrenophila (ATCCNo. 23309). Alternatively, new attenuated Aeromonas strains can beconstructed by introducing one or more attenuating mutations in groups(i) to (vii) as described for Shigella spp. above.

The particular Franciesella strain employed is not critical to thepresent invention. Examples of Franciesella strains which can beemployed in the present invention include F. tularensis (ATCC No.15482). Attenuated Franciesella strains are preferably used in thepresent invention, and can be constructed by introducing one or moreattenuating mutations in groups (i) to (vii) as described for Shigellaspp. above.

The particular Corynebacterium strain employed is not critical to thepresent invention. Examples of Corynebacterium strains which can beemployed in the present invention include C. pseudotuberculosis (ATCCNo. 19410). Attenuated Corynebacterium strains are preferably used inthe present invention, and can be constructed by introducing one or moreattenuating mutations in groups (i) to (vii) as described for Shigellaspp. above.

The particular Citrobacter strain employed is not critical to thepresent invention. Examples of Citrobacter strains which can be employedin the present invention include C. freundii (ATCC No. 8090). AttenuatedCitrobacter strains are preferably used in the present invention, andcan be constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Chlamydia strain employed is not critical to the presentinvention. Examples of Chlamydia strains which can be employed in thepresent invention include C. pneumoniae (ATCC No. VR1 310). AttenuatedChlamydia strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Hemophilus strain employed is not critical to the presentinvention. Examples of Hemophilus strains which can be employed in thepresent invention include H. sornmis (ATCC No. 43625). AttenuatedHemophilus strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Brucella strain employed is not critical to the presentinvention. Examples of Brucella strains which can be employed in thepresent invention include B. abortus (ATCC No. 23448). AttenuatedBrucella strains are preferably used in the present invention, and canbe constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Mycobacterium strain employed is not critical to thepresent invention. Examples of Mycobacterium strains which can beemployed in the present invention include M. intracelhilare (ATCC No.13950) and M. tuberculosis (ATCC No. 27294). Attenuated Mycobacteriumstrains are preferably used in the present invention, and can beconstructed by introducing one or more attenuating mutations in groups(i) to (vii) as described for Shigella spp. above.

The particular Legionella strain employed is not critical to the presentinvention. Examples of Legionella strains which can be employed in thepresent invention include L. pneumophila (ATCC No. 33156). AttenuatedLegionella strains, such as a L. pneumophila mip mutant (Ott, FEMSMicro. Rev., 14:161-176 (1994)) are preferably used in the presentinvention. Alternatively, new attenuated Legionella strains can beconstructed by introducing one or more attenuating mutations in groups(i) to (vii) as described for Shigella spp. above.

The particular Rhodococcus strain employed is not critical to thepresent invention. Examples of Rhodococcus strains which can be employedin the present invention include R. equi (ATCC No. 6939). AttenuatedRhodococcus strains are preferably used in the present invention, andcan be constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Pseudomonas strain employed is not critical to thepresent invention. Examples of Pseudomonas strains which can be employedin the present invention include P. aeruginosa (ATCC No. 23267).Attenuated Pseudomonas strains are preferably used in the presentinvention, and can be constructed by introducing one or more attenuatingmutations in groups (i) to (vii) as described for Shigella spp. above.

The particular Helicobacter strain employed is not critical to thepresent invention. Examples of Helicobacter strains which can beemployed in the present invention include H. mustelae (ATCC No. 43772).Attenuated Helicobacter strains are preferably used in the presentinvention, and can be constructed by introducing one or more attenuatingmutations in groups (i) to (vii) as described for Shigella spp. above.

The particular Salmonella stain employed is not critical to the presentinvention. Examples of Salmonella strains which can be employed in thepresent invention include Salmonella typhi (ATCC No. 7251) and S.typhimurium (ATCC No. 13311). Attenuated Salmonella strains arepreferably used in the present invention and include S. typhi aroC aroD(Hone et al Vacc, 9:810-816 (1991)) and S. typhimurium aroA mutant(Mastroeni et al. Micro. Pathol, 13:477-491 (1992))). Alternatively, newattenuated Salmonella strains can be constructed by introducing one ormore attenuating mutations in groups (i) to (vii) as described forShigella spp. above. The particular Vibrio strain employed is notcritical to the present invention.

Examples of Vibrio strains which can be employed in the presentinvention include Vibrio cholerae (ATCC No. 14035) and Vibriocincinnatiensis (ATCC No. 35912). Attenuated Vibrio strains arepreferably used in the present invention and include V. cholerae RSIvirulence mutant (Taylor et al J. Infect. Dis., 170:1518-1523 (1994))and V. cholerae ctxA, ace, zot, cep mutant (Waldor et al J. Infect.Dis., 170:278-283 (1994)). Alternatively, new attenuated Vibrio strainscan be constructed by introducing one or more attenuating mutations ingroups (i) to (vii) as described for Shigella spp. above.

The particular Bacillus strain employed is not critical to the presentinvention. Examples of Bacillus strains which can be employed in thepresent invention include Bacillus subtilis (ATCC No. 6051). AttenuatedBacillus strains are preferably used in the present invention andinclude B. anthracis mutant pX01 (Welkos et al Micro. Pathol, 14:381-388(1993)) and attenuated BCG strains (Stover et al Nat, 351:456-460(1991)). Alternatively, new attenuated Bacillus strains can beconstructed by introducing one or more attenuating mutations in groups(i) to (vii) as described for Shigella spp. above. The particularErysipelothrix strain employed is not critical to the present invention.

Examples of Erysipelothrix strains which can be employed in the presentinvention include Erysipelothrix rhusiopathiae (ATCC No. 19414) andErysipelothrix tonsillarum (ATCC No. 43339). Attenuated Erysipelothrixstrains are preferably used in the present invention and include E.rhusiopathiae Kg-Ia and Kg-2 (Watarai et al. J. Vet. Med. Sci.,55:595-600 (1993)) and E. rhusiopathiae ORVAC mutant (Markowska-Danielet al Int. J. Med. Microb. Viral. Parisit. Infect. Dis., 277:547-553(1992)). Alternatively, new attenuated Erysipelothrix strains can beconstructed by introducing one or more attenuating mutations in groups(i) to (vii) as described for Shigella spp. above.

2.3. Methods for Increasing the Invasive Properties of a BacterialStrain

Whether organisms have been traditionally described as invasive ornon-invasive, these organisms can be engineered to increase theirinvasive properties, e.g., by mimicking the invasive properties ofShigella spp., Listeria spp., Rickettsia spp., Salmonella spp, orenteroinvasive E. coli spp. For example, one or more genes that enablethe microorganism to access the cytoplasm of a cell, e.g., a cell in thenatural host of said non-invasive bacteria, can be introduced into themicroorganism.

Examples of such genes referred to herein as “cytoplasm-targeting genes”include genes encoding the proteins that enable invasion by Shigella orthe analogous invasion genes of entero-invasive Escherichia, orlisteriolysin O of Listeria, as such techniques are known to result inrendering a wide array of invasive bacteria capable of invading andentering the cytoplasm of animal cells (Formal et al. Infect. Immun.,46:465 (1984); Bielecke et al. Nature, 345:175-176 (1990); Small et al.In: Microbiology-1986, pages 121-124, Levine et al. Eds., AmericanSociety for Microbiology, Washington, D.C. (1986); Zychlinsky et al.Molec. Micro., 11:619-627 (1994); Gentschev et al (1995) Infection &Immunity 63:4202; Isberg, R. R. and S. Falkow (1985) Nature 317:262; andIsberg, R. R. et al. (1987) Cell 50:769). Methods for transferring theabove cytoplasm-targeting genes into a bacterial strain are well knownin the art. Another preferred gene which can be introduced into bacteriato increase their invasive character encodes the invasin protein fromYersinia pseudotuberculosis, (Leong et al. EMBO J., 9:1979 (1990)).Invasin can also be introduced in combination with listeriolysin,thereby further increasing the invasive character of the bacteriarelative to the introduction of either of these genes. The above geneshave been described for illustrative purposes; however, it will beobvious to those skilled in the art that any gene or combination ofgenes, from one or more sources, that participates in the delivery of amolecule, in particular an RNA or RNA-encoding DNA molecule, from amicroorganism into the cytoplasm of a cell, e.g., an animal cell, willsuffice. Thus, such genes are not limited to bacterial genes, andinclude viral genes, such as influenza virus hemagglutinin HA-2 whichpromotes endosmolysis (Plank et al. J. Biol. Chem., 269: 12918-12924(1994)). The above cytoplasm-targeting genes can be obtained by, e.g.,PCR amplification from DNA isolated from an invasive bacterium carryingthe desired cytoplasm-targeting gene. Primers for PCR can be designedfrom the nucleotide sequences available in the art, e.g., in theabove-listed references and/or in GenBank, which is publicly availableon the internet (www.ncbi.nlm.nih.gov/). The PCR primers can be designedto amplify a cytoplasm-targeting gene, a cytoplasm-targeting operon, acluster of cytoplasm-targeting genes, or a regulon ofcytoplasm-targeting genes. The PCR strategy employed will depend on thegenetic organization of the cytoplasm-targeting gene or genes in thetarget invasive bacteria. The PCR primers are designed to contain asequence that is homologous to DNA sequences at the beginning and end ofthe target DNA sequence. The cytoplasm-targeting genes can then beintroduced into the target bacterial strain, e.g., by using Hfr transferor plasmid mobilization (Miller, A Short Course in Bacterial Genetics,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992);Bothwell et al. supra; and Ausubel et al. supra), bacteriophage-mediatedtransduction (de Boer, supra; Miller, supra; and Ausubel et al. supra),chemical transformation (Bothwell et al. supra; Ausubel et al. supra),electroporation (Bothwel et al. supra; Ausubel et al. supra; andSambrook, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and physical transformationtechniques (Johnston et al. supra; and Bothwell, supra). Thecytoplasm-targeting genes can be incorporated into lysogenicbacteriophage (de Boer et al Cell, 56:641-649 (1989)), plasmids vectors(Curtiss et al. supra) or spliced into the chromosome (Hone et al.supra) of the target strain.

In addition to genetically engineering bacteria to increase theirinvasive properties, as set forth above, bacteria can also be modifiedby linking an invasion factor to the bacteria. Accordingly, in oneembodiment, a bacterium is rendered more invasive by coating thebacterium, either covalently or non-covalently, with an invasion factor,e.g., the protein invasin, invasin derivatives, or a fragment thereofsufficient for invasiveness. In fact, it has been shown thatnon-invasive bacterial cells coated with purified invasin from Yersiniapseudotuberculosis or the carboxyl-terminal 192 amino acids of invasinare able to enter mammalian cells (Leong et al. EMBO J. 9:1979 (1990)).Furthermore, latex beads coated with the carboxyl terminal region ofinvasin are efficiently internalized by mammalian cells, as are strainsof Staphylococcus aureus coated with antibody-immobilized invasin(reviewed in Isberg and Trail van Nhieu Ann. Rev. Genet. 27:395 (1994)).Alternatively, a bacterium can also be coated with an antibody, variantthereof, or fragment thereof which binds specifically to a surfacemolecule recognized by a bacterial entry factor. For example, it hasbeen shown that bacteria are internalized if they are coated with amonoclonal antibody directed against an integrin molecule, e.g., α5β1,known to be the surface molecule with which the bacterial invasinprotein interacts (Isberg and Tran van Nhieu, supra). Such antibodiescan be prepared according to methods known in the art. The antibodiescan be tested for efficacy in mediating bacterial invasiveness by, e.g.,coating bacteria with the antibody, contacting the bacteria witheukaryotic cells having a surface receptor recognized by the antibody,and monitoring the presence of intracellular bacteria, according to themethods described above. Methods for linking an invasion factor to thesurface of a bacterium are known in the art and include cross-linking.

3. Target Cells

The present invention provides a method for delivering RNA (orRNA-encoding DNA) to any type of target cell, where the RNA is miRNA ormiRNA precursor. As used herein, the term “target cell” refers to a cellwhich can be invaded by a bacterium, i.e., a cell which has thenecessary surface receptor for recognition by the bacterium.

Preferred target cells are eukaryotic cells. Even more preferred targetcells are animal cells. “Animal cells” are defined as nucleated,non-chloroplast containing cells derived from or present inmulticellular organisms whose taxonomic position lies within the kingdomanimalia. The cells may be present in the intact animal, a primary cellculture, explant culture or a transformed cell line. The particulartissue source of the cells is not critical to the present invention. Therecipient animal cells employed in the present invention are notcritical thereto and include cells present in or derived from allorganisms within the kingdom animalia, such as those of the familiesmammalia, pisces, avian, reptilia.

Preferred animal cells are mammalian cells, such as humans, bovine,ovine, porcine, feline, canine, goat, equine, and primate cells. Themost preferred animal cells are human cells.

In a preferred embodiment, the target cell is in a mucosal surface.Certain enteric pathogens, e.g., E. coli, Shigella, Listeria, andSalmonella, are naturally adapted for this application, as theseorganisms possess the ability to attach to and invade host mucosalsurfaces (Kreig et al. supra). Therefore, in the present invention, suchbacteria can deliver RNA molecules (miRNA or precursors) or RNA-encodingDNA to cells in the host mucosal compartment.

Although certain types of bacteria may have a certain tropism, i.e.,preferred target cells, delivery of RNA or RNA-encoding DNA to a certaintype of cell can be achieved by choosing a bacterium which has a tropismfor the desired cell type or which is modified such as to be able toinvade the desired cell type. Thus, e.g., a bacterium could begenetically engineered to mimic mucosal tissue tropism and invasiveproperties, as discussed above, to thereby allow said bacteria to invademucosal tissue, and deliver RNA or RNA-encoding DNA to cells in thosesites.

Bacteria can also be targeted to other types of cells. For example,bacteria can be targeted to erythrocytes of humans and primates bymodifying bacteria to express on their surface either, or both of, thePlasmodium vivax reticulocyte binding proteins-1 and -2, which bindspecifically to erythrocytes in humans and primates (Galinski et al.Cell, 69: 1213-1226 (1992)). In another embodiment, bacteria aremodified to have on their surface asialoorosomucoid, which is a ligandfor the asilogycoprotein receptor on hepatocytes (Wu et al. J. Biol.Chem., 263:14621-14624 (1988)). In yet another embodiment, bacteria arecoated with insulin-poly-L-lysine, which has been shown to targetplasmid uptake to cells with an insulin receptor (Rosenkranz et al.Expt. Cell Res., 199:323-329 (1992)). Also within the scope of thepresent invention are bacteria modified to have on their surface p60 ofListeria monocytogenes, which allows for tropism for hepatocytes (Hesset al. Infect. Immun., 63:2047-2053 (1995)), or a 60 kD surface proteinfrom Trypanosoma cruzi which causes specific binding to the mammalianextra-cellular matrix by binding to heparin, heparin sulfate andcollagen (Ortega-Barria et al. Cell, 67:411-421 (1991)).

Yet in another embodiment, a cell can be modified to become a targetcell of a bacterium for delivery of RNA. Accordingly, a cell can bemodified to express a surface antigen which is recognized by a bacteriumfor its targeted entry into the cell, i.e., a receptor of an invasionfactor. The cell can be modified either by introducing into the cell anucleic acid encoding a receptor of an invasion factor, such that thesurface antigen is expressed in the desired conditions. Alternatively,the cell can be coated with a receptor of an invasion factor. Receptorsof invasion factors include proteins belonging to the integrin receptorsuperfamily. A list of the type of integrin receptors recognized byvarious bacteria and other microorganisms can be found, e.g., in Isbergand Tran Van Nhieu Ann. Rev. Genet. 27:395 (1994). Nucleotide sequencesfor the integrin subunits can be found, e.g., in GenBank, publiclyavailable on the internet.

As set forth above, yet other target cells include fish, avian, andreptilian cells. Examples of bacteria which are naturally invasive forfish, avian, and reptilian cells are set forth below.

Examples of bacteria which can naturally access the cytoplasm offishcells include, but are not limited to Aeromonas salminocida (ATCC No.33658) and Aeromonas schuberii (ATCC No. 43700). Attenuated bacteria arepreferably used in the invention, and include A. salmonicidia vapA(Gustafson et al. J. Mol. Blot, 237:452-463 (1994)) or A. salmonicidiaaromatic-dependent mutant (Vaughan et al. Infect. Immun., 61:2172-2181(1993)).

Examples of bacteria which can naturally access the cytoplasm of aviancells include, but are not restricted to, Salmonella galinarum (ATCC No.9184), Salmonella enteriditis (ATCC No. 4931) and Salmonella typhimurium(ATCC No. 6994). Attenuated bacteria are preferred to the presentinvention and include attenuated Salmonella strains such as S. galinarumcya crp mutant (Curtiss et al. (1987) supra) or S. enteritidis aroAaromatic-dependent mutant CVL30 (Cooper et al. Infect. Immun.,62:4739-4746 (1994)).

Examples of bacteria which can naturally access the cytoplasm ofreptilian cells include, but are not restricted to, Salmonellatyphimurium (ATCC No. 6994). Attenuated bacteria are preferable to thepresent invention and include, attenuated strains such as S. typhimuirumaromatic-dependent mutant (Hormaeche et al. supra).

The present invention also provides for delivery of miRNA or itsprecursors to other eukaryotic cells, e.g., plant cells, so long asthere are microorganisms which are capable of invading such cells,either naturally or after having been modified to become invasive.Examples of microorganisms which can invade plant cells includeAgrobacterium tumerfacium, which uses a pilus-like structure which bindsto the plant cell via specific receptors, and then through a processthat resembles bacterial conjugation, delivers at least some of itscontent to the plant cell.

Set forth below are examples of cell lines to which RNA can be deliveredaccording to the method of this invention.

Examples of human cell lines include but are not limited to ATCC Nos.CCL 62, CCL 159, HTB 151, HTB 22, CCL 2, CRL 1634, CRL 8155, HTB 61, andHTB104.

Examples of bovine cell lines include ATCC Nos. CRL 6021, CRL 1733, CRL6033, CRL 6023, CCL 44 and CRL 1390. Examples of ovine cells linesinclude ATCC Nos. CRL 6540, CRL 6538, CRL 6548 and CRL 6546.

Examples of porcine cell lines include ATCC Nos. CL 184, CRL 6492, andCRL 1746.

Examples of feline cell lines include CRL 6077, CRL 6113, CRL 6140, CRL6164, CCL 94, CCL 150, CRL 6075 and CRL 6123.

Examples of buffalo cell lines include CCL 40 and CRL 6072.

Examples of canine cells include ATCC Nos. CRL 6213, CCL 34, CRL 6202,CRL 6225, CRL 6215, CRL 6203 and CRL 6575.

Examples of goat derived cell lines include ATCC No. CCL 73 and ATCC No.CRL 6270.

Examples of horse derived cell lines include ATCC Nos. CCL 57 and CRL6583.

Examples of deer cell lines include ATCC Nos. CRL 6193-6196.

Examples of primate derived cell lines include those from chimpanzee'ssuch as ATCC Nos. CRL 6312, CRL 6304, and CRL 1868; monkey cell linessuch as ATCC Nos. CRL 1576, CCL 26, and CCL 161; orangautan cell lineATCC No. CRL 1850; and gorilla cell line ATCC No. CRL 1854.

4. Pharmaceutical Compositions

In a preferred embodiment of the invention, the invasive bacteriacontaining the miRNA or its precursor molecules, and/or DNA encodingsuch, are introduced into an animal by intravenous, intramuscular,intradermal, intraperitoneally, peroral, intranasal, intraocular,intrarectal, intravaginal, intraosseous, oral, immersion andintraurethral inoculation routes.

Bacteria of the present invention can be lyophilized or made into theirspore form.

The amount of the live bacteria of the present invention to beadministered to a subject will vary depending on the species of thesubject, as well as the disease or condition that is being treated.Generally, the dosage employed will be about 10³ to 10¹⁵ viableorganisms, preferably about 10⁴ to 10¹² viable organisms per subject.

The invasive bacteria of the present invention are generallyadministered along with a pharmaceutically acceptable carrier and/ordiluent. The particular pharmaceutically acceptable carrier an/ordiluent employed is not critical to the present invention. Examples ofdiluents include a phosphate buffered saline, buffer for bufferingagainst gastric acid in the stomach, such as citrate buffer (pH 7.0)containing sucrose, bicarbonate buffer (pH 7.0) alone (Levine et al. J.Clin. Invest, 79:888-902 (1987); and Black et al, J. Infect. Dis.,155:1260-1265 (1987)), or bicarbonate buffer (pH 7.0) containingascorbic acid, lactose, and optionally aspartame (Levine et al. Lancet,11:467-470 (1988)). Examples of carriers include proteins, e.g., asfound in shin milk, sugars, e.g., sucrose, or polyvinylpyrrolidone.Typically these carriers would be used at a concentration of about0.1-30% (w/v) but preferably at a range of 1-10% (w/v).

Set forth below are other pharmaceutically acceptable carriers ordiluents which may be used for delivery specific routes. Any suchcarrier or diluent can be used for administration of the bacteria of theinvention, so long as the bacteria are still capable of invading atarget cell. In vitro or in vivo tests for invasiveness can be performedto determine appropriate diluents and carriers. The compositions of thepresent invention can be formulated for a variety of types ofadministration, including systemic and topical or localizedadministration. Lyophilized forms are also included, so long as thebacteria are invasive upon contact with a target cell or uponadministration to the subject. Techniques and formulations generally maybe found in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the composition, e.g., bacteria, of the present inventioncan be formulated in liquid solutions, preferably in physiologicallycompatible buffers such as Hank's solution or Ringer's solution.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner.

For administration by inhalation, the pharmaceutical compositions foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the composition, e.g., bacteria, and asuitable powder base such as lactose or starch.

The pharmaceutical compositions may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The pharmaceutical compositions may also be formulated in rectal,intravaginal or intraurethral compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides. Systemic administration can also beby transmucosal or transdermal means. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, for transmucosal administration bilesalts and fusidic acid derivatives. In addition, detergents may be usedto facilitate permeation. Transmucosal administration may be throughnasal sprays or using suppositories. For topical administration, thebacteria of the present invention are formulated into ointments, salves,gels, or creams as generally known in the art, so long as the bacteriaare still invasive upon contact with a target cell.

The compositions may, if desired, be presented in a pack or dispenserdevice and/or a kit which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

The invasive bacteria containing the miRNA/precursors or their encodingDNA to be introduced can be used to infect animal cells that arecultured in vitro, such as cells obtained from a subject. These invitro-infected cells can then be introduced into animals, e.g., thesubject from which the cells were obtained initially, intravenously,intramuscularly, intradermally, or intraperitoneally, or by anyinoculation route that allows the cells to enter the host tissue. Whendelivering RNA to individual cells, the dosage of viable organisms toadminister will be at a multiplicity of infection ranging from about 0.1to 10⁶, preferably about 10¹ to 10⁴ bacteria per cell.

In yet another embodiment of the present invention, bacteria can alsodeliver RNA molecules encoding proteins to cells, e.g., animal cells,from which the proteins can later be harvested or purified. For example,a protein can be produced in a tissue culture cell.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference, but they are not admitted tobe prior art to presently claimed invention. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES Material and Method Plasmid

For single promoter plasmid, the transkingdom miRNA (TMIR) plasmid wasconstructed as follows (FIG. 2). Briefly, annealed oligonucleotidescontaining multiple cloning site (MCS), T7 promoter and enhancer(synthesized by Qiagen) were ligated into blunted BssHII sites ofKSII(+). A DNA sequence encoding the human Let-7 miRNA (Let-7a) or oneof its precursors was inserted into the BamHI and SalI sites of MCS togenerate the plasmid pT7miRNA. The Let-7 miRNA family has been found toregulate the Ras gene (Johnson, S. et al, Cell, Vol. 120, 635-64792005)).

The Hly A gene was amplified from pGB2S2inv-hly (provided by C.Grillot-Courvalin) by PCR (Pfx DNA polymerase, Invitrogen Inc.) with thefollowing primers:

(SEQ ID NO: 1) hly-1: 5′-CCCTCCTTTGATTAGTATATTCCTATCTTA-3′, and(SEQ ID NO: 2) hly-2: 5′-AAGCTTTTAAATCAGCAGGGGTCTTTTTGG-3′,and were cloned into the EcoRV site of KSII(+). PstI fragmentscontaining the inv locus of pGB2Ωinv-hly were inserted into the PstIsite of KSII(+)/Hly. The Hly-Inv fragment was excised with BamHI andSail. After blunting, it was ligated into the EcoRV site incorporatedwithin the T7 terminator of pT7miRNA. The resulting TMIR plasmidencoding the Let-7a miRNA or one of its precursors was transformed intoE. coli (BL21 (DE3) pLysE) cells. Cells were cultured overnight to allowfor expression and miRNA processing.

For double-promoter plasmid, the T7-Therapeutic Pathway Identificationand Validation (TPIV®) plasmid was constructed to include an RNAicassette with two T7 promoters (FIG. 3). The desired DNA molecule iscloned into the plasmid through the two XbaI sites using standardmolecular cloning techniques. The plasmid also includes the Inv locusand Hly locus similar to the TMIR plasmid.

For vector construction, T7 Terminators were annealed according tostandard molecular biology techniques, and digested with eitherBamHIH/XbaI or XbaI/SalI. Terminators were then cloned into theBamHI/XbaI or XbaI/SalI sites of the TPIV® vector using the followingprimers:

BamHI sense: (SEQ ID NO: 3) 5′ACGGATCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGTCTAGAGGATC CAC 3′ BamHI antisense:(SEQ ID NO: 4) 5′ GTGGATCCTCTAGACCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGATC CGT 3′ SalI sense:(SEQ ID NO: 5) 5′ GCGTCGACTCTAGACCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGTCGA CCG 3′ SalI antisense:(SEQ ID NO: 6) 5′ CGGTCGACTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGTCTAGAGTCGA CGC 3′miRNA Assays

Hela cells were infected with E. coli (BL21 (DE3) pLysE) cells at an MOIof 1000 for 2 hours at 37° C. Twenty-four hours after infection,cellular RNA was harvested with TRIZOL (Invitrogen) and proteinextracted in lysis buffer. Amplification of k-Ras mRNA by RT-PCR wasconducted using OneStep RT-PCR kit (Invitrogen) and the followingprimers to amplify a 278 base pair fragment of k-Ras mRNA:

5′-AGTACAGTGCAATGAGGGACCAGT (SEQ ID NO: 7) and5′-AGCATCCTCCACTCTCTGTCTTGT. (SEQ ID NO: 8)The resulting PCR product was stained and run on an agrose gel andvisualized according to standard molecular biology procedures (FIG. 4,left), including RT-PCR, western blot, and/or northern blot analysis.

Specifically, western blot analysis to determine k-Ras protein levelswas performed using 50 ug cell extract separated on a 12% SDS-PAGE gel.k-Ras protein levels were determined by incubation with monoclonal k-Rasantibody (SantaCruz) and visualized by enhanced chemiluminescence (GEBiosciences) (FIG. 4, right).

Bacteria-Mediated, miRNA-Based Gene Modulation Against k-Ras Example 1Bacterial Expression of pre-miRNA

A fragment of the human Let-7 miRNA (Let-7a) purchased from IntegratedDNA Technologies, Inc. was cloned into the TMIR plasmid as describedabove.

The Let-7a DNA flanked by restriction enzyme sites (BamHI/SalI) has thefollowing sequence (SEQ ID NO:9):

5′-GCGGATCCTGGGATGAGGTAGTAGGTTGTATAGTTTTAGGGTCACACCCACCACTGGGAGATAACTATACAATCTACTGTCTTTCCTAGTCGAC CG-3′

Results of the mRNA assays are shown in FIG. 4. While the k-Ras mRNAlevel showed only slight decrease in cells treated with the Let-7a miRNAcompared to the control (left), the decrease in k-Ras protein level wasdisproportionally pronounced (right), suggesting that while some mRNAdegradation might have occurred, most of the gene modulation effect bythe Let-7a miRNA was likely through translation repression, a hallmarkof miRNA mechanism. These data clearly demonstrate that bacteria canmediate gene modulation via miRNA mechanism, through synthesizing andprocessing miRNA precursors.

Example 2 Bacterial Expression of pri-miRNA

DNA sequence encoding pri-miRNA sequence of the human miR-155 miRNA iscloned into the TMIR plasmid. Bacterial transformation with the plasmidis carried out. In one example, processing of the pri-miRNA to pre-miRNAoccurs in the bacteria with the expression of human Drosha either in thesame TMIR plasmid as the pri-miRNA sequence or on a separate bacterialexpression vector. In another example, pri-miRNA processing occurs inthe infected eukaryotic cells without the need for bacterial Droshaexpression.

Hela cells are then infected with the transformed bacteria and Bach1mRNA and protein levels are analyzed using the assays described.

The miR-155 pri-miRNA sequence is as follows:

(SEQ ID NO: 10) 5′-GTGGCACAAACCAGGAAGGGGAAATCTGTGGTTTAAATTCTTTATGCCTCATCCTCTGAGTGCTGAAGGCTTGCTGTAGGCTGTATGCTGTTAATGCTAATCGTGATAGGGGTTTTTGCCTCCAACTGACTCCTACATATTAGCATTACAGTGTATGATGCCTGTTACTAGCATTCACATGGAACAAATTGCTGCCGTGGGAGGATGACAAAGAAGCATGAGTCACCCTGCTGGATAAACTTAGACTTCAGGCTTTATCATTTTTCAATCTGTTAATCATAATCTGGTCACTGGGATGTTCAACCTTAAACTAAGTTTTGAAAGTAAGG-3′

Example 3 Bacterial Expression of miRNA Duplex

DNA sequences encoding two strands of a human miR-155 miRNA duplex iscloned into the TPIV® plasmid. Bacterial transformation with the plasmidis carried out. Hela cells are then infected with the transformedbacteria and k-Ras mRNA levels are analyzed using the assays described.

The miR-155 miRNA duplex sequences are as follows:

FOR 5′-TTAATGCTAATCGTGATAGGGG-3′ (SEQ ID NO: 11)REV 5′-CCCCTATCACGATTAGCATTAA-3′ (SEQ ID NO: 12)

Example 4 Bacterial Expression of miRNA

DNA sequence encoding the human miR-155 miRNA sequence is cloned intothe TMIR plasmid. Bacterial transformation with the plasmid is carriedout. Bela cells are then infected with the transformed bacteria andBach1 mRNA and protein levels are analyzed using the assays described.

The miR-155 miRNA sequence is as follows:

5′-TTAATGCTAATCGTGATAGGGG-3′ (SEQ ID NO: 11)

Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1. A DNA vector that encodes at least a microRNA (miRNA) or a miRNAprecursor, wherein said miRNA is capable of modulating the expression ofat least one eukaryotic, prokaryotic, or viral target gene.
 2. Thevector of claim 1 wherein said miRNA precursor is a pri-miRNA.
 3. Thevector of claim 1 wherein said miRNA precursor is a pre-miRNA.
 4. Thevector of claim 1 wherein said vector encodes duplex miRNAs, whereinsaid two miRNA strands comprise a substantially complementary region. 5.The vector of claim 1 wherein said miRNA is a mature miRNA.
 6. Thevector of claim 1 further comprising a prokaryotic promoter.
 7. Thevector of claim 1 further comprising a eukaryotic promoter.
 8. Thevector of claim 1 wherein said at least one target gene is acancer-related gene.
 9. The vector of claim 1 further encodes an Hlygene.
 10. A bacterium comprising a microRNA (miRNA), a miRNA precursor,or a DNA molecule encoding at least said miRNA or said precursor,wherein said miRNA is capable of modulating the expression of at leastone eukaryotic, prokaryotic, or viral target gene.
 11. The bacterium ofclaim 10 wherein the precursor is pre-miRNA.
 12. The bacterium of claim10 wherein the precursor is pri-miRNA.
 13. The bacterium of claim 10wherein the bacterium is a live invasive bacterium.
 14. The bacterium ofclaim 10 wherein the bacterium is a derivate of a live invasivebacterium.
 15. The bacterium of claim 10 wherein the bacterium isnon-pathogenic and non-virulent.
 16. The bacterium of claim 10, whereinthe bacterium is an attenuated strain selected from the group consistingof Listeria, Shigella, Salmonella, E. coli, and Bifidobacteriae.
 17. Thebacterium of claim 10, wherein the bacterium is selected from the groupconsisting of Yersinia spp., Escherichia spp., Klebsiella spp.,Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp.,Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp.,Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp.,Pseiidomonas spp., Helicobacter spp., Salmonella spp., Vibrio spp.,Bacillus spp., Leishmania spp. and Erysipelothrix spp. which have beengenetically engineered to mimic the invasion properties of Shigellaspp., Listeria spp., Rickettsia spp., and enteroinvasive E. coli spp.18. The bacterium of claim 10 further comprising an enzyme or ribozymethat is capable of processing the precursor closer to a mature miRNA.19. The bacterium of claim 18 wherein the enzyme is an endonuclease. 20.The bacterium of claim 10 further comprising at least one of a bacterialRNase III, a Dicer, a Dicer-like enzyme, Drosha, and Pasha.
 21. Thebacterium of claim 10 further comprising an enzyme that assists intransporting genetic materials, upon their release from the bacterium,into the cytoplasm of a target eukaryotic cell.
 22. The bacterium ofclaim 21 wherein said enzyme is an Hly protein.
 23. The bacterium ofclaim 10 further comprising a prokaryotic promoter controlling theexpression of said DNA molecule.
 24. The bacterium of claim 23, whereinthe promoter is T7 promoter.
 25. The bacterium of claim 10, furthercomprising a eukaryotic promoter controlling the expression of said DNAmolecule.
 26. The bacterium of claim 10 wherein the DNA molecule furtherencodes an Hly gene.
 27. The bacterium of claim 10 wherein theeukaryotic gene is an animal gene.
 28. The bacterium of claim 10 whereinthe eukaryotic gene is mammalian or avian gene.
 29. The bacterium ofclaim 10 wherein the at least one target gene is a cancer-related gene.30. A method of delivering a microRNA (miRNA) or a miRNA precursor to ananimal cell, said method comprising infecting said animal cell with abacterium comprising a microRNA (miRNA), a miRNA precursor, or a DNAmolecule encoding at least said miRNA or said precursor, wherein saidmiRNA is capable of modulating the expression of at least oneeukaryotic, prokaryotic, or viral target gene.
 31. The method of claim30, wherein said animal cell is a human cell.
 32. The method of claim30, further comprising lysing said bacterium after infecting. 33-47.(canceled)